GDNF ELISA Kit

Catalog No. ABIN625302

Product Details GDNF ELISA Kit

Target: GDNF (Glial Cell Line Derived Neurotrophic Factor (GDNF))

Reactivity: Human

Detection Method: Colorimetric

Method Type: Sandwich ELISA

Detection Range: 4-2000 pg/mL

Minimum Detection Limit: 4 pg/mL

Application: ELISA

Purpose: Human GDNF ELISA Kit for cell culture supernatants, plasma, and serum samples.

Sample Type: Plasma, Cell Culture Supernatant, Serum

Analytical Method: Quantitative

Specificity: This ELISA kit shows no cross-reactivity with the following cytokines tested: human Angiogenin, BDNF, BLC, ENA-78, FGF- 4, IL-1 alpha, IL-1 beta, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-11, IL-12 p70, IL-12 p40, IL-13, IL-15, I-309, IP-10, G-CSF, GM-CSF, IFN-gamma, Leptin (OB), MCP-1, MCP-2, MCP-3, MDC, MIP-1 alpha, MIP-1 beta, MIP-1 delta, PARC, PDGF, RANTES, SCF, TARC, TGF-beta, TIMP-1, TIMP-2, TNF-alpha, TNF-beta, TPO, VEGF.

Sensitivity: < 4 pg/mL

Characteristics:

• Strip plates and additional reagents allow for use in multiple experiments
• Quantitative protein detection
• Establishes normal range
• The best products for confirmation of antibody array data

Components:

• Pre-Coated 96-well Strip Microplate
• Wash Buffer
• Stop Solution
• Assay Diluent(s)
• Lyophilized Standard
• Biotinylated Detection Antibody
• Streptavidin-Conjugated HRP
• TMB One-Step Substrate

Material not included:

• Distilled or deionized water
• Precision pipettes to deliver 2 μL to 1 μL volumes
• Adjustable 1-25 μL pipettes for reagent preparation
• 100 μL and 1 liter graduated cylinders
• Tubes to prepare standard and sample dilutions
• Absorbent paper
• Microplate reader capable of measuring absorbance at 450nm
• Log-log graph paper or computer and software for ELISA data analysis

Application Details

Application Notes: Recommended Dilution for serum and plasma samples2 fold

Sample Volume: 100 μL

Plate: Pre-coated

Protocol:

1. Prepare all reagents, samples and standards as instructed in the manual.
2. Add 100 μL of standard or sample to each well.
3. Incubate 2.5 h at RT or O/N at 4 °C.
4. Add 100 μL of prepared biotin antibody to each well.
5. Incubate 1 h at RT.
6. Add 100 μL of prepared Streptavidin solution to each well.
7. Incubate 45 min at RT.
8. Add 100 μL of TMB One-Step Substrate Reagent to each well.
9. Incubate 30 min at RT.
10. Add 50 μL of Stop Solution to each well.
11. Read at 450 nm immediately.

Reagent Preparation:

1. Bring all reagents and samples to room temperature (18 - 25 °C) before use.
   2. Sample dilution: If your samples need to be diluted, Assay Diluent A (Item D) should be used for dilution of serum/plasma samples. 1x Assay Diluent B (Item E) is used for dilution of culture supernantants and urine. Suggested dilution for normal serum/plasma: 2 fold. Please note that levels of the target protein may vary between different specimens. Optimal dilution factors for each sample must be determined by the investigator.
   3. Assay Diluent B should be diluted 5-fold with deionized or distilled water before use.
   4. Preparation of standard: Briefly spin the vial of Item C and then add 400 µL Assay Diluent A (for serum/plasma samples) or 1x Assay Diluent B (for cell culture supernates/urine) into Item C vial to prepare a 50 ng/mL standard. Dissolve the powder thoroughly by a gentle mix. Add 20 µL GDNF standard (50 ng/mL) from the vial of Item C, into a tube with 480 µL Assay Diluent A or 1x Assay Diluent B to prepare a 2,000 pg/mL standard solution. Pipette 400 µL Assay Diluent A or 1x Assay Diluent B into each tube. Use the 2,000 pg/mL standard solution to produce a dilution series . Mix each tube thoroughly before the next transfer. Assay Diluent A or 1x Assay Diluent B serves as the zero standard (0 pg/mL). 20 µL standard + 480 µL 200 µL 200 µL 200 µL 200 µL 200 µL 200myl 2,000 666.7 222.2 74.07 24.69 8.23 2.74 0 pg/mL pg/mL pg/mL pg/mL pg/mL pg/mL pg/mL pg/mL
   5. If the Wash Concentrate (20x) (Item B) contains visible crystals, warm to room temperature and mix gently until dissolved. Dilute 20 ml of Wash Buffer Concentrate into deionized or distilled water to yield 400 ml of 1x Wash Buffer.
   6. Briefly spin the Detection Antibody vial (Item F) before use. Add 100 µL of 1x Assay Diluent B into the vial to prepare a detection antibody concentrate. Pipette up and down to mix gently (the concentrate can be stored at 4 °C for 5 days). The detection antibody concentrate should be diluted 80-fold with 1x Assay Diluent B and used in step 4 of Part VI Assay Procedure.
   7. Briefly spin the HRP-Streptavidin concentrate vial (Item G) and pipette up and down to mix gently before use. HRP-Streptavidin concentrate should be diluted 400-fold with 1x Assay Diluent B. For example: Briefly spin the vial (Item G) and pipette up and down to mix gently . Add 25 µL of HRP-Streptavidin concentrate into a tube with 10 ml 1x Assay Diluent B to prepare a final 400 fold diluted HRP- Streptavidin solution (don't store the diluted solution for next day use). Mix well.

Assay Procedure:

1. Bring all reagents and samples to room temperature (18 - 25 °C) before use. It is recommended that all standards and samples be run at least in duplicate.
   2. Add 100 µL of each standard (see Reagent Preparation step 2) and sample into appropriate wells. Cover well and incubate for 2.5 hours at room temperature or over night at 4 °C with gentle shaking.
   3. Discard the solution and wash 4 times with 1x Wash Solution. Wash by filling each well with Wash Buffer (300 myl) using a multi-channel Pipette or autowasher. Complete removal of liquid at each step is essential to good performance. After the last wash, remove any remaining Wash Buffer by aspirating or decanting. Invert the plate and blot it against clean paper towels.
   4. Add 100 µL of 1x prepared biotinylated antibody (Reagent Preparation step 6) to each well. Incubate for 1 hour at room temperature with gentle shaking.
   5. Discard the solution. Repeat the wash as in step
   6. Add 100 µL of prepared Streptavidin solution (see Reagent Preparation step 7) to each well. Incubate for 45 minutes at room temperature with gentle shaking.
   7. Discard the solution. Repeat the wash as in step
   8. Add 100 µL of TMB One-Step Substrate Reagent (Item H) to each well. Incubate for 30 minutes at room temperature in the dark with gentle shaking.
   9. Add 50 µL of Stop Solution (Item I) to each well. Read at 450 nm immediately.

Calculation of Results:

Calculate the mean absorbance for each set of duplicate standards, controls and samples, and subtract the average zero standard optical density. Plot the standard curve on log-log graph paper or using Sigma plot software, with standard concentration on the x-axis and absorbance on the y-axis. Draw the best-fit straight line through the standard points.
Typical Data: These standard curves are for demonstration only. A standard curve must be run with each assay. Assay Diluent A Human GDNF concentration (pg/mL) 1 10 100 1000 10000 O D =4 50 n m 0.001 0.01 0.1 1 10 Assay Diluent B Human GDNF concentration (pg/mL) 1 10 100 1000 10000 O D =4 50 n m 0.01 0.1 1 10
Sensitivity: The minimum detectable dose of GDNF is typically less than 4 pg/mL.
Recovery: Recovery was determined by spiking various levels human GDNF into human serum, plasma and cell culture media. Mean recoveries are as follows: Sample Type Average % Recovery Range ( %) Serum 102.9 92-114 Plasma 99.25 86-113 Cell culture media 119.8 85-140
Linearity: Sample Type Serum Plasma Cell Culture Media 1:2 Average % of Expected 77.70 106.6 102.1 Range ( %) 70-90 89-132 92-110 1:4 Average % of Expected 76.53 104.3 101.3 Range ( %) 68-91 88-132 90-109
Reproducibility: Intra-Assay: CV<10 % Inter-Assay: CV<12 %

Assay Precision: Intra-Assay: CV< 10 % Inter-Assay: CV< 12 %

Restrictions: For Research Use only

Handling

Handling Advice: Avoid repeated freeze-thaw cycles.

Storage: -20 °C

Storage Comment: The entire kit may be stored at -20°C for up to 1 year from the date of shipment. Avoid repeated freeze-thaw cycles. The kit may be stored at 4°C for up to 6 months. For extended storage, it is recommended to store at -80°C.

Expiry Date: 6 months

References for GDNF ELISA Kit (ABIN625302)

Faroni, Smith, Lu, Reid: "Human Schwann-like cells derived from adipose-derived mesenchymal stem cells rapidly de-differentiate in the absence of stimulating medium." in: The European journal of neuroscience, Vol. 43, Issue 3, pp. 417-30, (2016) (PubMed).

Wu, Li, Li, Lin, Yu, Luan, Ma: "Transport of glial cell line-derived neurotrophic factor into liposomes across the blood-brain barrier: in vitro and in vivo studies." in: International journal of molecular sciences, Vol. 15, Issue 3, pp. 3612-23, (2014) (PubMed).

Ye, Dang, Viet, Dolan, Schmidt: "Analgesia targeting IB4-positive neurons in cancer-induced mechanical hypersensitivity." in: The journal of pain : official journal of the American Pain Society, Vol. 13, Issue 6, pp. 524-31, (2012) (PubMed).

Kalbermatten, Schaakxs, Kingham, Wiberg: "Neurotrophic activity of human adipose stem cells isolated from deep and superficial layers of abdominal fat." in: Cell and tissue research, Vol. 344, Issue 2, pp. 251-60, (2011) (PubMed).

Whone, Kemp, Sun, Wilkins, Scolding et al.: "Human bone marrow mesenchymal stem cells protect catecholaminergic and serotonergic neuronal perikarya and transporter function from oxidative stress by the secretion of glial-derived neurotrophic ..." in: Brain research, Vol. 1431, pp. 86-96, (2011) (PubMed).

Thomas, Cullum, Siahamis, Langlois: "Infrared thermographic imaging, magnetic resonance imaging, CT scan and myelography in low back pain." in: British journal of rheumatology, Vol. 29, Issue 4, pp. 268-73, (1990) (PubMed).

Target Details for GDNF

Target: GDNF (Glial Cell Line Derived Neurotrophic Factor (GDNF))

Alternative Name: GDNF (GDNF Products)

Synonyms: gdnf ELISA Kit, GDNF ELISA Kit, AI385739 ELISA Kit, gdnfa ELISA Kit, hfb1-gdnf ELISA Kit, ATF1 ELISA Kit, ATF2 ELISA Kit, HFB1-GDNF ELISA Kit, HSCR3 ELISA Kit, glial cell derived neurotrophic factor ELISA Kit, glial cell derived neurotrophic factor a ELISA Kit, glial cell line derived neurotrophic factor ELISA Kit, glial cell derived neurotrophic factor L homeolog ELISA Kit, Gdnf ELISA Kit, gdnfa ELISA Kit, GDNF ELISA Kit, gdnf.L ELISA Kit

Background: The Human GDNF (Glial Derived Neurotrophic Factor) ELISA (Enzyme-Linked Immunosorbent Assay) kit is an in vitro enzyme-linked immunosorbent assay for the quantitative measurement of human GDNF in serum, plasma, cell culture supernatants and urine. This assay employs an antibody specific for human GDNF coated on a 96-well plate. Standards and samples are pipetted into the wells and GDNF present in a sample is bound to the wells by the immobilized antibody. The wells are washed and biotinylated anti-human GDNF antibody is added. After washing away unbound biotinylated antibody, HRP-conjugated streptavidin is pipetted to the wells. The wells are again washed, a TMB substrate solution is added to the wells and color develops in proportion to the amount of GDNF bound. The Stop Solution changes the color from blue to yellow, and the intensity of the color is measured at 450 nm. Reproducibility: Intra-Assay: CV<10% Inter-Assay: CV<12%.

Gene ID: 2668

UniProt: P39905

Pathways: RTK Signaling, Synaptic Membrane, Tube Formation, Autophagy, Smooth Muscle Cell Migration